1. Field of the Invention
The invention relates generally to the field of phosphorus chemistry, and is particularly concerned with a novel method for the production of sulfur-containing phosphonoformates and their derivatives. An additional aspect of the present invention relates to the use of these compounds as antiviral agents, including: Human Immunodeficiency Virus (HIV); herpes viruses including, Herpes Simplex Virus (HSV), Ebstein-Barr Virus (EBV), Varicella Zoster Virus (VZV), Cytomegalovirus (CMV), HSV-6, and HSV-8 (Kaposi""s sarcoma); Human Papilloma Virus (HPV); rhinoviruses; and hepatitis-linked viruses.
2. Description of Related Art
A retrovirus designated HIV is believed to be the causative agent of the complex disease termed Acquired Immune Deficiency Syndrome (AIDS). The complex disease AIDS includes progressive destruction of the immune system and degeneration of the central and peripheral nervous system. The HIV virus appears to preferentially attack helper T-cells (T-lymphocytes or OKT4-bearing T-cells) and also other human cells, e.g., certain cells within the brain. The helper T-cells are invaded by the virus and the T-cells become an HIV virus producer. The helper T-cells are quickly destroyed and their number in the human being is depleted to such an extent that the body""s B-cells as well as other T-cells normally stimulated by the helper T-cells no longer function normally or produce sufficient lymphokines and antibodies to destroy the invading virus or other invading microbes.
While the HIV virus does not necessarily cause death per se, it does cause severe damage to the human immune system resulting in the onset of various other opportunistic diseases such as, herpes, toxoplasmosis, cytomegalovirus (CMV), Kaposi""s sarcoma, and EBV related lymphomas, among others, (AIDS). HIV virus infected individuals, at first, experience few or no symptoms. Later in the disease, the onset of immune system dysfunction occurs, leading to various symptoms such as weight loss, malaise, fever, and swollen lymph nodes (AIDS related complex). The disease further progresses to full blown AIDS, leading usually to death. Those infected with the HIV virus are persistently infective to others.
Unfortunately, the present state of the art is such that antiviral drugs are only capable of attacking such viruses when they are replicating. Attacking a latent virus such as HIV, which does not reproduce itself following infection until reactivated by presently unknown factors would require distinguishing the viral genetic material from the surrounding host genetic material and selectively destroying it. Thus, the current generation of antiviral drugs is only effective against replicating viruses. Considerable efforts are being directed toward the control of HIV by means of inhibition of the reverse transcriptase of HIV and of other targets of viral functions, required for replication of the virus. Unfortunately, many of the known compounds suffer from toxicity problems, lack of bioavailability or are short-lived in vivo, viral resistance, or combinations thereof. Particularly important is combined therapy agents targeting more than one viral function. The requirement for development of new drugs able to be used in combination with other types of drugs is important in continued HIV therapy.
Several researchers have indicated that the pyrophosphonate analogues, such as phosphonoformic acid (PFA) and its analogues and derivatives possess antiviral properties in that they inhibit the replication of several viruses. (See, U.S. Pat. Nos. 5,072,032 and 5,183,812 to McKenna; D. W. Hutchinson, et al., Synthesis and Biochemical Properties of Some Pyrophosphate Analogues, Biophosphates and Their Analogues-Synthesis, Structure, Metabolism and Activity, K. S. Bruzik and W. J. Stec (Eds.), Elsevier Science Publishers, B. V., 1987, 441-450;. and Helgstrands, et al., Science, 201:819-821 (1978)). Trisodium phosphonoformate (PFA, Foscarnet), a pyrophosphate analog, has been reported to inhibit HIV reverse transcriptase (HIV, RT) with an ID50 near 1 xcexcm, and has also been reported to inhibit several herpes virus DNA polymerases, including the DNA polymerase of CMV. See, U.S. Pat. No. 5,072,032 to McKenna.
Previous studies reported the addition of nucleophiles to the thiocarbonyl group. (See, J. Levillain, et al., J. Am. Chem. Soc. 115, 8444-8446, 1993; and L. V. Kovalenko, et al., Russian J. General Chemistry 64, Part 1, 1456-1459, 1994). Preparations of thiocaibonyl and dithiocarboxyl derivatives have been demonstrated in the reaction of trialkyl phosphate and chlorothioformate. (See, D. W. Grisley, Jr., J. Org. Chem. 26, 2544-2546, 1961.) However, trimethyl phosphonoformate thio derivatives have not been previously reported, except trimethyl thiophosphonoformate, U.S. Pat. No.5,072,032 to C. E. McKenna, et al., and trimethyl phosphonothionoformate, Kovalenko et al., supra. Corresponding monosodium salts and trisodium salts are, disclosed for the first time, except for the monosodium and trisodium salts of thiophosphonoformate which were previously disclosed McKenna, et al, supra.
The present invention solves the above-described problems by providing methods for readily synthesizing sulfur-containing phosphonoformic acid derivatives and the use of such derivatives as chemotherapeutic agents. In one embodiment of the invention, sulfur-containing phosphonoformate derivatives are obtained formally by replacing one or more of the five oxygen atoms of the original phosphonoformate molecule by a sulfur atom. Another embodiment of the present invention is a method for synthesizing polyhydroxy derivatives of such analogs. The method uses the base compound (foscarnet), but can be extended to prepare similar derivatives based on the thio analogues of foscarnet. An additional aspect of the present invention relates to the use of these compounds as antiviral agents, including: HIV and herpes viruses.
These and other features, aspects, and advantages of the present invention will become better understood with regard to the following detailed description and appended claims.
The present invention teaches the synthesis of sulfur-containing phosphonoformic acid derivatives, which are obtained by replacing one or more of the five oxygen atoms of the original phosphonoformate molecule by a sulfur atom, see TABLE I. CAS is the Chemical Abstract Service.
General synthetic pathways as applied to examples of various thio-analogs of PFA are outlined in the following conceptual schemes: 
The unique biological activities of the compounds disclosed herein, and their corresponding classes, derive chiefly from two factors: 1) modification of their reactivity, cell transport, cell permeation, metabolism, and enzyme or membrane receptor site binding properties due to the different chemical and physical properties of sulfur relative to oxygen; and 2) potential in situ physiological conversion of S to O, creating the possibility of prodrugs for which the actual drug has one or more S converted to O after administration. Another factor is the modified properties of prodrugs or other analogues in which the sulfur-containing function is derivatized, e.g. as an ester, ether, etc., relative to metabolic activation in vivo.
The analogues have the general structure: 
R1 and R2 are each independently selected from alkyl, aryl, H, or cation, R3 is independently selected from alkyl, aryl, H, or cation:
X1 X2, X3, X4, and X5 are O or S, provided that:
(a) at least one of X1xe2x88x92X5 is S;
(b) when X1 is S, then either (i) R1 or R2 is alkyl, aryl, or H, or (ii) at least one of X2, X3, X4, and X5 is also S.
The parent structures may form part of a derived entity wherein R1, R2 and/or R3 are more complex molecules than simple alkyl or aryl compounds (or portions of the same molecule), with the parent incorporated via one or more esteratic or ether bonds as indicated above. The cation can be a pharmaceutically acceptable alkali metal (e.g., Li, Na, or K), ammonium cation; alkaline earth cation (e.g., Ca2+, Ba2+, Mg2+), higher valency cation, or polycationic counter ion (e.g., a polyamonium cation). See, Berge, et al., xe2x80x9cPharmaceutical Saltsxe2x80x9d, J. Pharm. Sci. (1977) 66:1-19. It will be appreciated that the stoichiometry of an anionic compound to a salt-forming counterion (if any) will vary depending on the charge of the anionic portion of the compound (if any) will vary depending on the charge of the anionic portion of the compound (if any) and the charge of the counterion. Preferred pharmaceutically acceptable salts include a sodium, potassium or calcium salt, but other salts are also contemplated within their pharmaceutically acceptable range. Furthermore R1, R2, and R3 may be so designed as to create novel biologically active compounds or prodrugs, wherein one conjugating moiety may be for example a nucleoside or nucleotide with independent activity, and another moiety may be for example a diol, triol or higher polyhydroxy group conferring enhanced cell transport or other desirable properties.
Classes of Parent Structures (X1xe2x88x92X5=O Except as Noted)
The following compilation sets forth permutations for illustrative purposes. It will be appreciated that simple experiments by those skilled in the art will readily eliminate those compounds that are not stable or are synthetically unattainable.
In accordance with this invention, TPFA is conjugated with polyalcohols. We postulate that such compounds can have enhanced membrane transport properties, and thus higher activity than the parent in vivo. Some 15 years ago, oral delivery of different classes of drugs was shown to be facilitated by incorporation of 1-O-alkyl, 1-O-acyl-sn-glycerol-phosphate moieties. See, Ryu, et al., J. Med. Chem. 25, 1322-1329 (1982). More recent examples of this approach have been given by M. Fuji,.et al., J. Org. Chem., 62, 6804 (1997) and by K. Hostetler, et al., Antiviral Research, 31, 59-67 (1996).
Our proposed synthetic routes are illustrated in the examples below. 
Wherein R1 is alkyl, such as C3H7 or C16H33; R2 is alkyl, such as CH3; and Y is 0 or S.
General Experimental Protocol
Solvents for reactions were purified as follows: THF was distilled from sodium and benzophenone ketyl, toluene was distilled from CaH2, and acetone was dried with molecular sieve. Solvents for column chromatography or Thin Layer Chromatography (TLC) were not pretreated. The end points of all reactions were checked using TLC or NMR except noted. Monosodium salts and trisodium salts were dried in a vacuum, and all the products were stored at 4xc2x0 C. R1 is an alkyl group having 1 to 22 carbon atoms. R2 is an alkyl group having 1 to 6 carbon atoms. X is a halogen, such as chlorine, bromine, and iodine.
All solvents and reagents were of Analytical Reagent (AR) grade quality, purchased from. Sigma-Aldrich,. Inc., without further purification except where noted. Nuclear Magnetic Resonance (NMR) spectra were recorded on CDCl3 solutions for triesters and on D2O for monosodium salts and trisodium salts. 1H and 13C spectra were recorded on Bruker AC250 MHz or AM360 MHz spectrometers, in CDCl3. 1H chemical shifts are referenced to CHCl3 (xcex47.24). 13C chemical shifts were referenced to: CDCl3 (xcex477.0); D2O HDO (xcex44.63); and C6D6 (xcex4128). 31P spectra were recorded on the 360 MHz instrument. 31P NMR chemical shifts are referenced to external 85% H3PO4. Chemical shifts are reported in ppm (s=singlet, d=doublet, t-triplet, q=quartet, m=multiplet). High Resolution Mass Spectra (HRMS) determinations were performed at UC Riverside.